Smallpox vaccines were used in the past all over the world and contributed to the eradication of smallpox. However, all vaccine strains at that time were problematic in that 1) the strains induced serious adverse effects such as postvaccinal encephalitis and 2) it was difficult to guarantee their sterility because vaccine production was dependent on a primitive technique that involves inoculating a virus into bovine skin and then extracting the resultant from the thus-formed abscess. To address such problems, Hashizume et al., at the Chiba Serum Institute passaged a Lister strain (thought to cause relatively fewer adverse effects compared with other smallpox vaccine strains used all over the world at that time) in primary rabbit kidney (PRK) cells at 30° C. and then established an LC16 strain from the resultant as a temperature-sensitive strain that does not proliferate at temperatures of 40.8° C. or higher. The LC16 strain had greatly attenuated pathogenicity against the monkey central nervous system compared with that of the parental Lister strain, or other strains used as vaccines at that time. However, it was revealed that the proliferation property of the LC16 strain was rather elevated in rabbit skin. Accordingly, passage culture was further continued in PRK cells, so that an LC16mO strain (hereinafter also referred to as an mO strain) that forms smaller pocks was selected. Approximately 1,000 people were vaccinated with the mO strain. However, even the mO strain induce strong dermal reactions, and an LC16m8 strain (hereinafter also referred to as an m8 strain) was established as a strain that forms even smaller-sized pocks from the mO strain (So Hashizume, Clinical Virology, Vol. 3, No. 3, Jul. 1, 1975). 100,000 infants were vaccinated with the m8 strain from 1974 to 1975 without any serious adverse effects being reported. With its safety demonstrated to be higher than that of conventional vaccine strains, the m8 strain was approved as an official vaccine strain by the Ministry of Health and Welfare in Japan. Despite its weak ability to proliferate in skin compared with the mO strain, the m8 strain exhibited antibody-inducing ability at almost the same level as that of its parental Lister strain. Furthermore, the m8 strain was clearly better in terms of safety than the mO strain. Another major improvement of the m8 strain was that the strain can be produced by aseptic tissue culture using primary cultured rabbit kidney cells. However, based on applied research using the m8 strain and experience of actual production at the Chiba Serum Institute in 2001, it was shown that large plaque-forming revertants (reverse mutants) emerge during the m8 strain culture processes. It was revealed that the emergence of such revertant virus is an inevitable property of the m8 strain, since revertants also emerge from plaque-purified m8. It was also revealed that contamination with revertants may cause a concern regarding vaccine safety, since revertants' properties closely resemble those of the mO strain and their ability to proliferate in skin is elevated.
Moreover, because vaccinia viruses have a wide host range and high expression efficiency, the viruses have been used as vectors after introduction of other foreign genes (JP Patent Publication (Kohyo) No. 11-509091 A (1999)). The above LC16mO strain or LC16m8 strain has also been examined concerning its use as a vector because of its high safety.
However, as described above, the LC16mO strain is problematic in terms of property of proliferating in skin and the LC16m8 strain is problematic in terms of emergence of revertants. Thus, it has been required to generate a viral strain with higher safety as a smallpox vaccine strain or a vector virus.